NUCLEIC ACID FACILITY

NNUUCCLLEEIICC AACCIIDD FFAACCIILLIITTYY

CUSTOM OLIGONUCLEOTIDE SYNTHESIS

1999

Cancer Center

Department of Chemistry

University of Pennsylvania

Nucleic Acid Facility Phone: (215) 898-1584 Fax: (215) 573-2159 Web: www.med.upenn.edu/dna

2

Welcome to the NUCLEIC ACID FACILITY

The Nucleic Acid Facility has provided high quality custom oligonucleotides to the university community for the

past 15 years. Oligonucleotides from the facility are desalted and lyophilized, ready for use in PCR, sequencing,

cloning, mutagenesis and many other experiments. Customer satisfaction is guaranteed.

In addition to the standard oligonucleotide synthesis, the facility offers ready made standard primers (HPLC purified)

at low cost and the facility can furnish modified DNA or RNA for special needs. Among the special modifications

provided routinely are: phosphorothioate oligonucleotide, methyl phosphonate DNA, 2'-OMe methyl phosphonate

RNA, phosphorylation, cyclic oligonucleotides, oligonucleotide linkers, insertion of modified nucleosides into

specific sequences, and labeling with digoxigenin, biotin, cholesterol, DNP, acridine, psoralen, dyes, and dual dyes.

We welcome inquiries about specialty synthesis items not covered in this catalog. Contact us for free consultation

about your DNA application.

Custom oligonucleotides may be ordered online or by FAX and Email. Charges vary according to services. Cancer

Center members receive a 10% discount on all services. Other non-profit and commercial organizations are invited

to inquire about pricing. There is no charge for on-campus delivery. The charge for Federal Express shipment is

$10. Delivery times range from two days to two weeks, depending on the level of purification and modification

required.

Welcome

Location: 337 Cret Chemistry Building, 231 South 34th St.Philadelphia, PA 19104-6323

Technical Director: Xiaolin Zhang, Ph.D.Technicians: Michael Imbalzano

Dorothy HunterFacility Director: Ponzy Lu, Ph.D. Professor of Chemistry

Phone: (215) 898-1584Fax: (215) 573-2159Web: http://www.med.upenn.edu/dnaEmail: [email protected]


3

Overview of DNA Synthesis .......................................................................................................................................4

Overview of DNA Purification ................................................................................................................................5-6

Standard Oligonucleotide Synthesis

DNA Synthesis …….………………………………………………………………………..……………..7

RNA Synthesis ……………………………………………………………………………..……………..7

2'-OMe RNA Synthesis ………………………………………………….………………..………….…...7

Chimeric Oligonucleotide .............................................................................................................................7

Standard Primers ..........................................................................................................................................................8

Special Modifications

Phosphorothioate Oligonucleotide ………………….……………….…………………………….……….9

Methyl Phosphonate DNA ………………………….……………………………………..……….….…..9

2'-OMe Methyl Phosphonate RNA................................................................................................................9

Phosphorylation ……………………………………....………………………………..……………....….10

Cyclic Oligonucleotide.................................................................................................................................10

Oligonucleotide Linkers ………………….……………....………………………………………….…....11

Digoxigenin Labeling .................................................................................................................................12

Biotin Labeling ………...…...……...….…………….…………………………………………….…..….12

Cholesterol Labeling ...................................................................................................................................13

DNP Labeling ..............................................................................................................................................13

Acridine Labeling ........................................................................................................................................14

Psoralen Labeling ........................................................................................................................................14

Dye Labeling ……………............……………………………….……….......…………....……..…........15

Dual Dye Labeling ......................................................................................................................................16

Modified Nucleosides ……...…...........……………………….……………………………………....17-19

Order Form.................................................................................................................................................................20

Table of Contents


4

The field of DNA synthesis exploded with the pioneering work of Caruthers and his coworkers in the early 1980s.Caruthers' team is credited with the development of silica-based solid supports and the discovery of the highlyefficient nucleoside phophoramidite synthesis reagents. Phosphoramidites later led to the development of the firsttruly successful automated DNA synthesizers, having a major impact in the fields of molecular biology,biotechnology, and biological chemistry.

DNA synthesis is a cyclical process that assembles a chain of nucleotides from the 3'-end to the 5'-end. The 3'-nucleoside is covalently attached to a solid support and successive nucleotide monomers are added one by onethrough a cycle of four chemical reactions: detritylation, coupling, capping and oxidation. As the followingillustration shows, the first step of the synthesis cycle is detritylation, where the dimethoxytrityl (DMT) group isremoved with trichloroacetic acid to free the 5' hydroxyl for the coupling reaction. The next step is coupling, inwhich the 5'-OH of the oligonucleotide reacts with an activated monomer created by simultaneously adding thephosphoramidite nucleoside monomer and tetrazole. The tetrazole protonates the nitrogen of the phosphoramidite,making it susceptible to nucleophilic attack. The next step, capping, terminates any chains that did not undergocoupling. Since the unreacted chains have a free 5' OH, they are capped by acetylation with acetic anhydride and 1-methylimidazole. Capping minimizes the length of impurities, making it easy to do post-synthesis trityl-selectivepurification of the final product. Finally, the internucleotide linkage is converted from the phosphite to the morestable phosphotriester by oxidation with an iodine solution. For the synthesis of phosphorothioate oligonucleotides,the internucleotide phosphite is oxidized by a sulfuring reagent. After oxidation, the DMT group is removed withtrichloroacetic acid and the cycle is repeated until chain elongation is complete. The amount of DMT released fromeach cycle is monitored to insure high coupling efficiency. The protected oligonucleotide with/without DMT iscleaved from the solid support with concentrated ammonium hydroxide. Ammonia treatment also removes thecyanoethyl phosphate protecting groups. The crude DNA in ammonium hydroxide solution is then heated to removethe protecting groups on the exocyclic amines of the bases.

Advances in nucleic acid chemistry have made possible the synthesis of oligonucleotides with modified backbones,non-standard bases, or with non-radioactive labels attached to the 3' or 5'-termini. RNA, OMe-RNA and othermodified oligonucleotides can be synthesized with corresponding monomer phosphoramidites by using the samesynthesis cycle as DNA.

All the oligonucleotides are synthesized on ExpediteTM 8909 Nucleic Acid Synthesis Systems in this facility.

Overview of DNA Synthesis

References:Agrawal, S. (1993). Protocols for Oligonucleotidesand Analogs, 20.

Agrawal S. (1994). Protocols for OligonucleotideConjugates, 26.

Beaucage, S. L. and Caruthers, M. H. (1981).Tetrahedron Lett., 22, 1859.

Eckstein, F. (1991). Oligonucleotides and Analogies.

Letsinger, R. L. finnan, J. L., Heavner, G. A., andLunsford, W. B. (1975). J. Am. Chem. Soc., 97, 3278.

McBride, L. J. and Caruthers, M. H. (1983).Tetrahedron Lett., 24, 245.

Matteucci, M. D. and Caruthers, M. H. (1980).Tetrahedron Lett. 21, 719.

DNA Synthesis Cycle

BO

DMTO

O

PO OCH2CH2CN

OO B

O

ODMTO

O

OHO

O

ODMTO

O

P OCH2CH2CN

OO B

O

ODMTO

O

PiPr2N OCH2CH2CN

Detritylation

OO

O

Coupling

Monomer

CH3C

Oxidation

O

Capping

B

Solid Support

B

B

B

B

Detritylation


5

Our advanced DNA synthesizers and our care with chemical reagents result in high quality oligonucleotides.Desalted DNA is suitable for direct use in sequencing or PCR in many cases. However, we offer a range of DNApurification protocols using tC-18 Sep-PakTM cartridge or reversed-phase HPLC. The recovery yield from oligopurification is 25-50% of the synthetic yield. DNA with modifications may give lower yields.

The step-wise coupling efficiency for DNA synthesis is about 99%. The final yield is dependent on the length and sequence of the oligo.

DESALT PURIFICATION

After deprotection, the ammonia solution is removed from the oligonucleotide by vacuum. All the protected groups,small failure strands and ammonia salt are then removed by sodium acetate-ethanol precipitation. The resulting oligois in sodium salt form. Desalted, quantified and lyophilized DNA, with synthesis report, is delivered in 2 businessdays.

The electropherogram of a desalted 48-mer: 5'GCATGACTGGTGGTCAGCAAAT The electropherogram of desalted 89-mer:5'GGAATTCCATATGAAAAGCCAGGGCCCTGAGGAAAACTCCCAATCCC TTTTGAACCACCTACCCTTCACGAACTGTATGATTTAGACGTGACGGCC

CCAAACCTTGTACCGGAGG

HPLC Purification

The trityl-on full length DNA strands are separated from the trityl-on and trityl-off failure strands by HPLC on areversed-phase PRP-1 Hamilton column (4.1 x 250 mm), and the trityl groups are removed on the column withtrifluoroacetic acid. The full length oligos are collected, desalted with ethanol precipitation, quantified andlyophilized. HPLC-purified DNA, with HPLC and synthesis report, is delivered in 5 business days.

The electropherogram of an HPLC-purified 20-mer: 5'GTCATCATCATCCG The electropherogram of an HPLC-purified 89-mer: 5'GGAATTCCATATGGGTCTC AAAAGCCATTTTGAACCACCTACCCTTCACGAACTGTATGATTTAGACG

TGACGGCCCCAAACCTTGTACCGGAGG

Synthesis Scale Desalted Yield (20-mer) HPLC Yield (20-mer)

25-nmole 5 OD 0.15 mg NA NA50-nmole 10 OD 0.30 mg 3 OD 0.01 mg

0.2-µmole 40 OD 1.20 mg 15 OD 0.45 mg1.0-µmole 160 OD 4.80 mg 60 OD 1.80 mg10-µmole 1600 OD 48.00 mg 600 OD 18.00 mg

Overview of DNA Purification


6

Cartridge Purification

The trityl-on DNA strands are separated from the trityl-off failure strands on a tC-18 Sep-PakTM cartridge.Detritylation is carried out on the cartridge with trifluoroacetic acid. Detrityled oligos are collected, desalted withethanol precipitation, quantified and lyophilized. Cartridge purification is less effective than HPLC andrecommended only for unmodified oligos less than 40 nucleotides in length. Cartridge purified DNA, with synthesisreport, is delivered in 3 business days.

The electropherogram of a cartridge-purified 21-mer: 5'TGAAGGATGGACAT The electropherogram of of a cartridge-purified 36-mer:GACGGAC 5'CAAACAGACACCATGGTTGTGCGGCGTATCTTCACC

QUALITY CONTROL

To ensure the oligonucleotides are of high quality, all the oligonucleotides are synthesized by highly trainedtechnicians using state of the art DNA synthesizers and high quality chemical reagents. The trityl color is monitoredto ensure high coupling efficiency for each oligonucleotide synthesized in this facility. Any oligonucleotide that arenot meet our standards will be remade. The quality of all the oligonucleotides with special modifications are checkedby capillary electrophoresis(CE). At least 10% of the DNA oligonucleotides are analyzed by CE routinely.

NOTES ABOUT DNA SAMPLESAll DNA samples are delivered as lyophilized powders and should be stored at -20°C. The DNA samples should bestored at -70°C if dissolved in water or buffer.

One OD unit is the absorbency of a 1.0 mL oligonucleotide solution, measured at 260 nm in a 1.0 cm path-lengthcuvette. One OD unit is approximately equal to 33 micrograms of single-stranded DNA, or 5 nanomoles of a 20-mer.

Overview of DNA Purification


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DNA Synthesis

DNA (up to 200 nucleotides) is synthesized on ExpediteTM 8909 Nucleic Acid Synthesis Systems using standardphosphoramidite chemistry. There are no set-up charges.

* Cancer Center member rate.

RNA Synthesis

RNA (up to 40 nucleotides) is synthesized on ExpediteTM 8909 Nucleic Acid Synthesis System with 2'-silyl protectedRNA phosphoramidites. Deprotected and desalted RNA oligos are delivered in 10 business days. There are no set-up charges. No purification is available for RNA oligos.

Yields of RNA oligo are greatly dependent on the sequence.

The coupling efficiency for RNA synthesis is about 95%.

2'-OMe RNA Synthesis2’-OMe RNA (up to 40 nucleotides) is synthesized with 2'-OMe RNA phosphoramidites. Deprotected and desaltedoligos are delivered in 10 business days. There are no set-up charges. HPLC purification is available for 2'-OMeRNA oligos (charge is same as DNA purification).

The coupling efficiency for 2’-OMe RNA synthesis is about 95% .

Chimeric Oligonucleotide Synthesis

Oligonucleotides containing a mixture of different sugars and/or backbone chemistries can be synthesized. Manypossible chimeric oligonucleotides can be synthesized. Please call for more details.

Synthesis Charge/base Purification Charge/oligoSynthesis Scale CC* PENN Cartridge HPLC

25-nmole $0.80 $0.90 NA NA50-nmole $0.90 $1.00 $10 $25

0.2-µmole $1.10 $1.20 $10 $251.0-µmole $2.50 $2.75 $10 $5010-µmole $14.00 $15.50 NA $200

Synthesis Scale Yield(20-mer) Charge/base0.2-µmole 15 OD $8.001.0-µmole 70 OD $10.00

Synthesis Scale Yield(20-mer) Charge/base HPLC/oligo0.2-µmole 20 OD $8.00 $251.0-µmole 80 OD $10.00 $50

Standard Oligonucleotide Synthesis

O

BASE

O

O OMe

P

O-

O

O

OO

BASE

OMe

2'-OMeRNA

BASE

OO

O BASE

OO

O

P

O-

O

OH

OH

RNA

BASE

OO

O BASE

OO

O

P

O-

O

DNA


8

All the primers listed below are HPLC purified and checked by CE to insure the high quality. For an updated list,please see our web site. If a common primer you require is not listed, it will be made available to you free of charge.

Primers are packaged in vials of 1 OD unit each; charge is $10/ primer. Same day delivery.

* Extinction Coefficient

Primers Sequence Length Tm (°°C) MW ε ∗ε ∗((OD/µµmol)T7 Promoter 5'-TAA TAC GAC TCA CTA TAG GG-3' 20-mer 51 6125 205T3 Promoter 5'-CAA TTA ACC CTC ACT AAA GG-3' 20-mer 51 6054 203M13 Forward (-20) 5'-GTA AAA CGA CGG CCA GTG-3' 18-mer 54 5558 188M13 Forward (-41) 5'-GGT TTT CCC AGT CAC GAC-3' 18-mer 54 5451 163M13 Reverse (-27) 5'-GGA AAC AGC TAT GAC CAT G-3' 19-mer 53 5846 200M13 Reverse (-48) 5'-AGC GGA TAA CAA TTT CAC AC-3' 20-mer 51 6094 207SP6 Promoter 5'-TAC GAT TTA GGT GAC ACT ATA G-3' 22-mer 53 6773 225pBluescript SK 5'-CGC TCT AGA ACT AGT GGA TC-3' 20-mer 55 6117 194pBluescript KS 5'-CTC GAG GTC GAC GGT ATC G-3' 19-mer 59 5854 180Lambda gt11 (forward) 5'-GGT GGC GAC GAC TCC TGG AGC CCG-3' 24-mer 71 7396 223Lambda gt11 (reverse) 5'-TTG ACA CCA GAC CAA CTG GTA ATG-3' 24-mer 59 7346 243Lambda gt10 (forward) 5'-AGC AAG TTC AGC CTG GTT AAG-3' 21-mer 56 6470 212Lambda gt10 (reverse) 5'-CTT ATG AGT ATT TCT TCC AGG GTA-3' 24-mer 55 7349 227pBR322 Bam HI, CW 5'-CAC TAT CGA CTA CGC GAT CA-3' 20-mer 55 6046 192pBR322 Bam HI, CCW 5'-ATG CGT CCG GCG TAG A-3' 16-mer 52 4922 155pBR322 Eco RI, CW 5'-GTA TCA CGA GGC CCT T-3' 16-mer 50 4857 148pBR322 Eco RI, CCW 5'-GAT AAG CTG TCA AAC-3' 15-mer 42 4584 158pBR322 HIND III, CW 5'-GAC AGC TTA TCA TCG-3' 15-mer 44 4552 145pBR322 HIND III, CCW 5'-GCA ATT TAA CTG TGA T-3' 16-mer 42 4895 162pBR322 Pst I, CW 5'-GCT AGA GTA AGT AGT T-3' 16-mer 44 4960 168pBR322 Pst I, CCW 5'-AAC GAC GAG CGT GAC-3' 15-mer 48 4611 156pBR322 Sal I, CW 5'-ATG CAG GAG TCG CAT-3' 15-mer 46 4617 152pBR322 Sal I, CCW 5'-AGT CAT GCC CCG CGC-3' 15-mer 52 4514 132Random Hexamer 5'-NNNNNN-3' 6-mer 58 1792 12PolydT20 5'-TTT TTT TTT TTT TTT TTT TT-3' 20-mer 35 6022 158

Standard Primers


9

Special modifications of oligonucleotides with commercially available phosphoramidite reagents can be performedin our facility. For information about any oligonucleotide modification not listed, please call Dr. Xiaolin Zhang.

Modified oligonucleotides are delivered in 10 business days. HPLC purification is recommended to ensure quality.

Phosphorothioate Oligonucleotide

Phosphorothioate analogues of DNA, RNA and OMe-RNA have sulphur in place of oxygen as one of the non-bridging ligands bonded to phosphorus. Thiolation of the oligo phosphates at any or all positions are possible at noextra charge. Purification is available using HPLC only.

Methyl Phosphonate DNA

DNA oligo containing one or more methyl phosphonate linkages can be synthesized. HPLC purification is available.

2'-OMe Methyl Phosphonate RNA

The 2'-OMe methyl phosphonate RNA has uncharged backbone linkages.Please call for more details.

Synthesis Scale Charge/base0.2-µmole $8.001.0-µmole $10.00


BASE

OO

O BASE

OO

O

P

O-

S

S-DNA

BASE

OO

O BASE

OO

O

P

O-

S

OH

OH

S-RNA

O

BASE

O

O OMe

P

O-

S

O

OO

BASE

OMe

S-OMeRNA

BASE

OO

O BASE

OO

O

P

CH3

O

Methyl phosphonate DNA

BASE

OO

O BASE

OO

O

P

CH3

O

2'-OMe Methyl Phosphonate RNA

OCH3

OCH3


10

PhosphorylationThe standard synthetic oligonucleotide has an OH group on both 5' and 3'-end. Phosphorylation at 5' and/or 3' end ofthe oligo is available. Price includes HPLC purification.

Cyclic Oligonucleotides

Cyclic oligonucleotides of length within the range of 2 to 20 nucleotides can be prepared. Please call for moredetails.

Phosphorylation 0.2-µmole/oligo 1.0-µmole/oligo5’-Phosphorylation $35 $65

3’-Phosphorylation $35 $70

O- BASE

OO

O

P

O-

O

5'-Phosphorylated oligo

BASE

OO

O

P

O-

O-O

3'-Phosphorylated oligo

HN

N

CH3

O

O

OO

O

P

O

O-O

O

O

BASE

P

O

O-O

O

O

BASE

P

O

O-

n

Cyclic Oligonucleotide

n<30-mer



11

Oligonucleotide Linkers

Oligonucleotide can be linked with different functional groups through a variety of hydrocarbon chains to fit thedesired applications. Price includes HPLC purification.

Oligonucleotide Linkers 200nmole/oligo 1.0-µmole/oligo5’-C3-Amino-oligo $35 $65

5’-C6-Amino-oligo $35 $655’-C12-Amino-oligo $45 $753’-C3-Amino-oligo $30 $603’-C6-Amino-oligo $30 $603’-C7-Amino-oligo $30 $60Amino-C2-dT-oligo inquire inquireAmino-C6-dT-oligo inquire inquireCarboxy-dT-oligo inquire inquire5’-HS-C6-oligo $40 $703’-HS-C3-oligo $35 $65Spacer 9-oligo $40 $70Spacer C3-oligo $40 $70Spacer 18-oligo $45 $75dSpacer-oligo $45 $753’-Spacer-C3-oligo $35 $70


O BASE

O

O

O

P

O-

OH2N

5'-C3-Amino-oligo

O BASE

O

O

O

P

O-

OH2N

5'-C6-Amino-oligo

O BASE

O

O

O

P

O-

O

5'-C12-Amino-oligo

H2N

BASE

OO

O

OP

O-

ONH2

OH

3'-C7-Amino-oligo

HSO P

O-

O

OBASE

O

O

5'-HS-C6-oligo

BASE

OO

O

OP

O-

O SH

3'-HS-C3-oligo

ON

O

O

NH

O

O O

NH

NH2

Amino Modifier-C2-dT-oligo

ON

O

O

NH

O

O O

NH

NH2

Amino Modifier-C6-dT-oligo

ON

O

O

NH

O

O O

OH

Carboxy-dT oligo

HOO

OO

OO

O

P

O-

O

BASE

Spacer 9-oligo

HO

O

OO

O

P

O-

O

BASE

Spacer C3-oligo

OO

O

dSpacer-oligo

BASE

OO

O

OP

O-

O OH

3'-Spacer-C3-oligo

HOO

O

O

OO

O

P

O-

O

BASE

Spacer 18-oligo

OO

O

BASE

O

O

O

OP

O-

O

NH2

3'-C6-Amino-oligo


12

Digoxigenin labeling

Price includes HPLC purification.

Biotin LabelingPrice includes HPLC purification. For multiple labeling, please call.

Digoxigenin labeling 200nmole/oligo 1.0-µmole/oligo

5'-Digoxigenin-oligo $100 $150

3'-Digoxigenin-oligo $80 $120

Biotinylation 200nmole/oligo 1.0-µmole/oligo5'-Biotin-oligo $40 $70

3’-Biotin-TEG-oligo $35 $70

Biotin-dT-oligo Internal labeling. Inquire

Biotin-TEG-oligo Internal labeling. Inquire


O BASE

OO

O

P

O-

OHN

O

S

NHHN

O

5'-Biotin-oligo

HN

O

S

HN NH

O

OO

O

N

HN

O

O

NH

O

Biotin-dT-oligo

BASE

OO

O

OP

O-

O OO

OO

HN

O

S

HN NH

O

OH

3'-Biotin-TEG-oligo

O BASE

OO

O

P

O-

O

Biotin-TEG-oligo

OH

OO

OO

HN

OS

NHHN

O

O

O

OH

CH3HO

CH3

HNH

OP-O

O

BASE

O

O

O

HN

O

OOH

3'-Digoxigenin-oligo

O

O

OH

CH3HO

CH3

HHN

BASE

O

O

OS

O P

O-

OO

5'-Digoxigenin-oligo


13

Cholesterol LabelingPotential therapeutic oligonucleotides must permeate the cell membrane for optimal activity. The addition oflipophilic groups to an oligonucleotide is expected to enhance activity. Price includes HPLC purification. Formultiple labeling, please call.

DNP LabelingOligonucleotide labeled with 2,4-dinitrophenyl (DNP) can be detected with anti-NDP antibodies. Price includesHPLC purification. For multiple labeling, please call.

Cholesterol Labeling 200nmole/oligo 1.0-µmole/oligo

5'-Cholesteryl-TEG-oligo $80 $120

3'-Cholestryl-TEG-oligo $35 $70

DNP Labeling 200nmole/oligo 1.0-µmole/oligo

5'-DNP-TEG-oligo $80 $120


OH

OO

OO N

HO

O

O

5'-Cholesteryl-TEG-oligo

OO

O

Base

P

O

O-

O2NHN

NO2

OO

OO

OH

O P

O

O

-OBASE

O

O

5'-DNP-TEG-oligo

O

OO

OO N

HO

OH

O

3'-Cholesteryl-TEG-oligo

O

O

BASEO

P O-

O


14

Acridine LabelingAcridine is an effective intercalating agent. Price includes HPLC purification. For multiple labeling, please call.

Psoralen LabelingPsoralen C2 placed at the 5' terminus of an oligonucleotide serves effectively as a cross-linking reagent in double-stranded oligonucleotides. Price includes HPLC purification. For multiple labeling, please call.

Acridine Labeling 200nmole/oligo 1.0-µmole/oligo

5'-Acridine-oligo $80 $120

3'-Acridine-oligo $35 $70

Psoralen Labeling 200nmole/oligo 1.0-µmole/oligo

5'-Psoralen-C2-oligo $70 $100

5'-Psoralen-C6-oligo $70 $100

N

MeO

Cl

HNOH

O

O BASE

OO

O

P

O-

5'-Acridine-oligo


N

MeO

Cl

HNO

OH

3'-Acridine-oligo

BASE

OHO

O

P O-

O

O OCH3

CH3

CH3

O

OO

O BASE

OO

O

P

O-

5'-Psoralen-C2-oligo

O OCH3

CH3

CH3

O

OO

O BASE

OO

O

P

O-

5'-Psoralen-C6-oligo


15

Dye LabelingOligonucleotide can be labeled with different dyes at either end and/or internally. The area of fluorescence detectionof DNA hybrids is developing rapidly, particularly in the areas of fluorescence in situ hybridization and in hybriddetection on DNA chips. Fluorescent labels provide for very sensitive detection. Under normal laboratory conditionsthe fluorescein-labeled oligo can detect 5x10-17 moles of target molecules. For any dye labeling not listed, please callthe facility. Price includes HPLC purification.

* Extinction Coefficient

Dye labeling 200nmole 1.0µmole ColorAbsorbance

(max) Emission

(max) ε ε (cm-1M-1)*

5’-Fluorescein-oligo $50 $80 Green 494nm 525nm 73,000

3’-Fluorescein-oligo $40 $70 Green 494nm 525nm 73,0005’-6-FAM-oligo $80 $120 Green 494nm 525nm 73,0005’-HEX-oligo $80 $120 Pink 535nm 556nm 73,0005’-TET-oligo $80 $120 Orange 521nm 536nm 73,000Fluorescein-dT-oligo inquire inquire Green 494nm 525nm 73,000TAMRA-dT-oligo $130 $180 Rose 565nm 580nm 89,0003’-TAMRA-oligo $40 $70 Rose 565nm 580nm 89,0005'-Cy3TM-oligo $80 $120 Orange 550nm 570nm 150,0005'-Cy5TM-oligo $80 $120 Far red 649nm 670nm 250,000DABCYL-dT-oligo $130 $180 -- 453nm none 32,0003’-DABCYL-oligo $50 $80 -- 453nm none 32,000


5'-Fluorescein-oligo

O

OO

O

P

O-

O

O OHO

COOH

HN

O

BASE

O

OO

O

P

O-

O

O OHO

COOH

HNBASEO

5'-6-FAM-oligo

NH

NH

O

CO2-

O

N

N+

O

HN

N

O

OO

O

O

TAMRA-dT-oligo

N N NNH

OHN

O

HN

NO

O

O

O

O

DABCYL-dT-oligo N N NNH

O

O

3'-DABCYL-oligo

HO

P

O

-O

O

OO

BASE

O BASE

OO

O

P

O-

O

N+N

HO

5'-Cy5TM -oligo

O BASE

OO

O

P

O-

O

N+N

HO

5'-Cy3TM -oligo

NH

NH

O

CO2-

O

OH

O

O

HN

N

O

OO

O

O

Fluorescein-dT-oligo

OH

ONH

O

CO2-

O

N

N+

BASE

OHO

O

OP

O-

O

3'-TAMRA-oligo

BASE

OO

O

OP

O-

ONH

OH

O OHO

COOH

NH

S

3'-Florescein-oligo

O

OO

O

P

O-

O

O OHO

COOH

HNBASEO

ClCl

Cl

Cl

5'-TET-oligo

O

OO

O

P

O-

O

O OHO

COOH

HNBASEO

ClCl

Cl

Cl

5'-HEX-oligo

Cl Cl


16

Dual Dye Labeling

In many case, it is desirable to label an oligo with more than one dye. Several current diagnostic assays utilizingoligo with two dyes-labeled that carries both a reporting and a quenching dye on opposite termini, such as TaqManprobe for the Perkin-Elmer TaqMan system and molecular beacon. We can finish those probes at low cost. Beforeordering, please call for more details. Price includes HPLC purification.

TaqManTM ProbeThe assay exploits the 5' nuclease activity of Taq DNA Polymerase to allow direct detection of the PCR product bythe release of the fluorescent reporter during PCR amplification. The TaqMan probe consists of an oligonucleotidewith a 5'-reporter dye (fluorescein, FAM or TET) and a 3'-quencher dye, TAMRA. The fluorescence of the reporteris quenched by the TAMRA dye. When the probe is intact, the proximity of the reporter dye to the quencher dyeresults in suppression of the reporter fluorescence. If hybridization occurs, probe cleavage by Taq polymerase takesplace during polymerization of the targeted amplicon, resulting in separation of the reporter and quencher, andcausing the reporter dye fluorescence to increase.

TaqMan probe with fluorescein as fluorophore and TAMRA as quencher

Molecular BeaconA molecular beacon probe has its natural fluorescence quenched in solution unless it is hybridized to the targetsequence. A molecular beacon contains a fluorophore at one terminus (such as fluorescein, FAM, HEX, TET,Edeans, Texas or others) and DABCYL as universal quencher at the other. The probe sequence is in the center of themolecule and the bases towards both termini are self-complementary and about 5-8 nucleotides in length dependingon the size of the probe sequence. The lengths of the probe sequences are chosen to maximize the separation of thefluorophore and quencher molecules when the probe is hybridized to the target.

Molecular Beacon with fluorescein as fluorophore and DABCYL as quencher.

Dual Dye Labeling 200nmole 1.0-µmole

TaqMan Probe $1.20/base of DNA +$150 $2.75/base of DNA +$300

Molecular Beacon $1.20/base of DNA +$150 $2.75/base of DNA +$300


OPO

-OO

OO

BASE

O

OO

O

PO-

O

O OHO

COOH

HN

BASE

5'-Fluorescein

oligo

3'-TAMRA

O

OHO

NH

O

CO2-

O

N

N+

N N NNH

OO

HO

PO

-OO

OO

BASE

O

OO

O

PO-

O

O OHO

COOH

HN

BASE

oligo

3'-DABCYL

O

5'-Fluorescein


17

Modified NucleosidesModified nucleosides can be incorporated into the oligonucleotide sequence at any sites.

Common Modified Nucleosides

Other Modified Nucleosides

Please call for more details.

2'-DeoxyAdenosine Analogs

Modified Nucleosides 200nmole/site 1.0-µmole/site2'-DeoxyInosine (dI) $8 $12

2'-DeoxyUridine (dU) $8 $122-Aminopurine $25 $355-Br-dC $15 $255-I-dC $15 $255-Br-dU $15 $255-I-dU $15 $255-Propynyl-dC $20 $305-Propynyl-dU $15 $25


O

N

NN

N

HO

OH

H2N

2-Aminopurine

N

N

NH2

O

OHO

OH

Br

5-Br-dC

N

N

NH2

O

OHO

OH

I

5-I-dC

O

N

NHN

N

HO

OH

O

2'-DeoxyInosine (dI)

N

HN

O

O

OHO

OH2'-DeoxyUridine (dU)

N

HN

O

O

OHO

OH

Br

5-Br-dU

N

HN

O

O

OHO

OH

I

5-I-dU

N

N

NH2

O

OHO

OH

CH3

5-Propynyl-dC

N

HN

O

O

OHO

OH

CH3

5-Propynyl-dU

O

N

NN

N

HO

OH

NHCH3

N6-Me-dA

O

N

N

N

HO

OH

NH2

7-Deaza-dA

O

N

NN

N

HO

OH

N

Etheno-dA

O

N

NN

N

HO

OHPurine

OHO

8-oxo-2'-deoxyAdenosine

N

HNN

N

OH

O

NH2

OHO

OH

7-Deaza-2'-deoxyAdenosine

N

N

N

NH2

OHO

OH

8-Bromo-2'-deoxyAdenosine

N

NN

N

NH2

Br

O

N

NN

NHO

OH

H2N

NH2

2,6-Diaminopurine


18

2'-DeoxyCytidine Analogs

2'-DeoxyGuanosine Analogs

Thymidine Analogs

Other 2'-Deoxyriboside Analogs


N

N

NH2

O

OHO

OH

CH3

5-Me-dC

N

N

NH2

O

OHO

OH

F

5-F-dC

N

N

NH2

O

OHO

OH

F

5-F-dC

O

N

HN

N

HO

OH

O

H2N

7-deaza-dG

O

N

NN

N

HO

OH

OCH3

H2N

O6-Me-dG

N

HN

O

O

OHO

OH

F

5-F-dU

N

N

OCH3

O

OHO

OH

CH3

O4-Me-T

OHO

OH

3'-Nitropyrrole

N

NO2

OHO

OH

dP

N

HN

ON

O

OHO

OH

5-M3IsodC

N

N

H2N

O

CH3

OHO

OH

IsodG

N

NN

NHO

NH2

OHO

OH

5-Nitroindole

N

NO2

OHO

OH

dK

N

NHN

NH2N

NOCH3

OHO

OH

2-Thio-dT

N

HN

S

CH3

O

N

N

NH2

H2N

OHO

OH

Cl

Diaminopyrazine-C-nucleoside

OI

5'-I-dT

N

OH

HN

O

O

CH3

OHO

5,6-Dihydro-dU

N

OH

HN

O

O

OHO

5,6-Dihydro-dT

N

OH

HN

O

O

CH3

H

OHO

OH

4-Thio-dT

N

HN

O

CH3

S

OHO

OH

4-Thio-dU

N

HN

O

S

OHO

8-oxo-2'-deoxyGuanosine

N

HNHN

N

OH

O

O

H2N

OHO

OH

2-Aminopurine

N

NN

NH2N

OHO

OH

8-Bromo-2'-deoxyGuanosine

N

NN

N

Br

O

H2N

OHO

4-Methylindole

N

OH

CH3

O

N

NHN

N

HO

OH

S

H2N

6-Thio-dG

OMeO

5'-OMe-dT

N

OH

HN

O

O

CH3

OH2N

5'-Amino-dT

N

OH

HN

O

O

CH3


19

3'-Deoxyriboside Analogs

RNA Analogs

2'OMe-RNA Analogs

N

HN

O

O

OHO

OH

Br

Br-UOH

N

HN

O

O

OHO

OH

I

I-U

OH

N

HN

O

OHO

OH

F

2'-OMe-5-F-UOCH3

O

OHO

2'-OMe-2-AP

N

NN

NH2N

OCH3OH

OHO

2'-OMe-2-amino-A

N

NN

NH2N

OCH3OH

NH2

OHO

2'-OMe-I

N

NHN

N

OCH3OH

O

N

N

O

OHO

OH2'-OMe-3-deaza-5-aza-C

OCH3

NH2

N

N

NH2

O

OHO

OH

CH3

2'-OMe-5-Me-COCH3

N

N

NH2

O

OHO

OH 2'-OMe-pC

OCH3

CH3

N

HN

O

O

OHO

OH 2'-OMe-pU

OCH3

CH3

N

HN

O

OHO

OH

CH3

2'-OMe-5-Me-UOCH3

O

N

HN

O

O

OHO

OH

2'-F-UF

N

HN

O

O

OHO

OH

2'-NH2-UNH2

OHO

3'-dC

N

N

NH2

O

OH

OHO

3'dA

N

OH

NN

N

NH2

OHO

3'dG

N

OH

NHN

N

O

H2N

OHO

3'dT

N

OH

HN

O

O

CH3

N

N

NH2

O

OHO

OH

Ara-C

OH

N

N

NH2

O

OHO

OH

2'-F-CF

N

N

NH2

O

OHO

OH

2'-NH2-CNH2


OHO

2'3'-ddC

N

N

NH2

O


20

NUCLEIC ACID FACILITYUniversity of Pennsylvania Cancer CenterSchool of Arts and Sciences, Department of Chemistry

337 Cret Chem Bldg., 231 S. 34th St., Philadelphia, PA 19104-5323Phone: (215) 898-1584 Fax: (215) 572-2159

Order online at www.med.upenn.edu/naf

Principal Investigator:_____________________________ Date: _/___/___

User:__________________________________________ Phone:____________

Account No.:____________________________________

Delivering Address (include department or school): Sequence Name:_________________________________________________________ Synthesis Scale:

_________________________________________ 25-nomle(no purification option)

_________________________________________ 50-nomle 0.2-µmole

Special Requirement:________________________ 1.0-µmole 10-µmole

_________________________________________ Purification: None

_________________________________________ Cartridge HPLC

Sequence(5' to 3'):

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

16 17 18 19 20 21 22 23 24 25 26 27 28 29 30

31 32 33 34 35 36 37 38 39 40 41 42 43 44 45

46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Order Form

Serial No.

IUB Mixed Base Symbols:B=(C,G,T/U) D=(A,G,T/U) H=(A,C,T/U) K=(G,T/U) M=(A,C) N=(A,C,G,T/U)R=(A,G) S=(C,G) V=(A,C,G) Y=(C,T/U) W=(A,T/U) X=Other

NUCLEIC ACID FACILITY

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